The nuclear pore complex (NPC) is the only known channel for macromolecular exchange between the nucleus and cytoplasm, and is therefore largely responsible for the establishment and maintenance of the unique properties of each compartment. In addition to the NPC itself, four soluble import factors are known to be involved in nuclear localization sequence (NLS) mediated import into the nucleus: karyopherins alpha and beta, Ran, and pl0. Binding between many of these purified proteins has been demonstrated in vitro. While these binding studies are informative, they do not directly address which of these import factors interact with each other during the actual import process, what the duration of these interactions is, or identify critical NPC proteins involved in the translocation process. Hence, the goals of these studies are two fold: (1) to ascertain the order and duration that these import factors associate with each other and with the NLS substrate during import, and (2) to identify additional components of the NPC that interact with the import substrate complex. First, crosslinking between import factors and NLS substrate will be used examine 2 distinct theories on the mechanism of nuclear import. Second, import factors joined to cross-linkers will reveal which import factors interact with each other during nuclear import. Third, crosslinkers attached to elongated NLS substrates will be used to probe for NPC proteins that closely associate with NLS substrates during transit through the NPC.